G-CSF is a glycoprotein having a molecular weight of about 20,000 and acting on precursor cells of neutrophils to promote their proliferation and differentiation to maturation.
Since we obtained high-purity human G-CSF by culturing a cell line collected from tumor cells of a patient with cancer of the floor of the mouth, the human G-CSF gene was successfully cloned and, at present, recombinant human G-CSF can be produced in mass in microorganisms or animal cells by genetic engineering techniques. We also succeeded in formulating this purified G-CSF into pharmaceutical products supplied to the market as antiinfection agents (Japanese Patent No. 2116515).
G-CSF is used in a very small amount, i.e., a formulation containing 0.1-1000 μg (preferably 5-500 μg) of G-CSF is normally administered once to seven times per week per adult. However, this G-CSF tends to be adsorbed to the walls of ampules for injection, syringes or the like. Moreover, G-CSF is unstable and susceptible to extrinsic factors such as temperature, humidity, oxygen, UV rays or the like to undergo physical or chemical changes including association, polymerization or oxidation, resulting in great loss of activity.
In order to prevent these influences, various formulation designs have been proposed mainly in dosage forms of freeze-dried formulations. For example, formulations containing at least one member selected from the group consisting of (a) at least one amino acid selected from threonine, tryptophan, lysine, hydroxylysine, histidine, arginine, cysteine, cystine and methionine; (b) at least one sulfur-containing reducing agent; or (c) at least one antioxidant were proposed (Japanese Patent No. 2577744). G-CSF formulations containing a surfactant such as a Polysorbate as a stabilizer were also proposed (JP-A-63-146826).
Freeze-dried G-CSF formulations containing maltose, raffinose, sucrose, trehalose or an aminosugar were also reported (JP-A-8-504784).
However, freeze-drying processes entail an increase in production costs on a commercial basis as well as an increase in the danger resulting from mechanical failure. Moreover, freeze-dried formulations had the problem that they must be dissolved in pure water (sterilized water for injection) before use.
Some products currently on the market contain a protein commonly used as a stabilizer such as human serum albumin or purified gelatin for controlling such chemical or physical changes. However, the addition of a protein as a stabilizer involved problems such as the necessity of a very complicated process for removing contamination with viruses.
However, production of oxidized G-CSF at methionine residues increases in the absence of such a protein, leading to deterioration.
For the reasons described above, there are demands for G-CSF solution formulations alternative to freeze-dried formulations, which are free from proteins as stabilizers and stable even after long-term storage.